Disruption of Abcc6 in the mouse: novel insight in the pathogenesis of pseudoxanthoma elasticum

Disruption of Abcc6 in the mouse: novel insight inthe pathogenesis of pseudoxanthoma elasticum

Theo G.M.F. Gorgels1, Xiaofeng Hu1, George L. Scheffer3, Allard C. van der Wal4,

Johan Toonstra5, Paulus T.V.M. de Jong1,6,8, Toin H. van Kuppevelt9, Christiaan N. Levelt2,

Anneke de Wolf1, Willem J.P. Loves1, Rik J. Scheper3, Ron Peek1 and Arthur A.B. Bergen1,7,*

1Department of Molecular and Clinical Ophthalmogenetics and 2Department Molecular Visual Plasticity, The

Netherlands Ophthalmic Research Institute (NORI), Amsterdam, The Netherlands, 3Department of Pathology, Free

University Amsterdam, 4Department of Cardiovascular Pathology, AMC, Amsterdam, 5Department of Dermatology,

UMC, Utrecht, The Netherlands, 6Department of Ophthalmology and 7Department of Clinical Genetics, AMC,

Amsterdam, The Netherlands, 8Department of Epidemiology and Biostatistics, EMC, Rotterdam, The Netherlands and9Department of Biochemistry, NCMLS, Medical Center, Radboud University Nijmegen, Nijmegen, The Netherlands

Received March 24, 2005; Revised and Accepted May 3, 2005

Pseudoxanthoma elasticum (PXE) is a heritable disorder of connective tissue, affecting mainly skin, eye andthe cardiovascular system. PXE is characterized by dystrophic mineralization of elastic fibres. The conditionis caused by loss of function mutations in ABCC6. We generated Abcc6 deficient mice (Abcc62/2) by conven-tional gene targeting. As shown by light and electron microscopy Abcc62/2 mice spontaneously developedcalcification of elastic fibres in blood vessel walls and in Bruch’s membrane in the eye. No clear abnormal-ities were seen in the dermal extracellular matrix. Calcification of blood vessels was most prominent in smallarteries in the cortex of the kidney, but in old mice, it occurred also in other organs and in the aorta and venacava. Newly developed monoclonal antibodies against mouse Abcc6 localized the protein to the basolateralmembranes of hepatocytes and the basal membrane in renal proximal tubules, but failed to show the proteinat the pathogenic sites. Abcc62 /2 mice developed a 25% reduction in plasma HDL cholesterol and anincrease in plasma creatinine levels, which may be due to impaired kidney function. No changes in serummineral balance were found. We conclude that the phenotype of the Abcc62/2 mouse shares calcificationof elastic fibres with human PXE pathology, which makes this model a useful tool to further investigatethe aetiology of PXE. Our data support the hypothesis that PXE is in fact a systemic disease.

INTRODUCTION

Pseudoxanthoma elasticum (PXE) is a heritable disorder ofthe connective tissue with multiple systemic manifestations.Clinical expression is variable and most frequently involvesskin, eye and blood vessels (1). Skin abnormalities consistof yellowish papules and plaques on the neck. Flexuralareas, such as axils and groin, develop redundant folds oflax skin (2). Examination of the fundus of the PXE eyereveals angioid streaks representing breaks in Bruch’smembrane, which may lead to choroidal neovascularization,haemorrhages and, consequently, loss of visual acuity (3).

Cardiovascular complications include hypertension, arterialinsufficiencies in the extremities and gastrointestinal haemor-rhages (3). Histopathological analysis of the affected tissuesreveals calcification and fragmentation of elastic fibres aswell as abnormalities in collagen fibrils (3,4). PXE symptomsusually appear in the second or third decade of life, althoughwe have seen patients with skin changes from 4 years on.The disease is progressive and as yet incurable.

The prevalence of PXE is at least one in 25 000 (5,6). Thevast majority of patients are sporadic cases. In familial cases,the disease almost exclusively segregates in an autosomalrecessive fashion (6). Recently, PXE was found to be caused

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*To whom correspondence should be addressed at: Department of Clinical and Molecular Ophthalmogenetics, The Netherlands Ophthalmic ResearchInstitute, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands. Tel: þ31 205666101; Fax: þ31 205666121; Email: [email protected]

Human Molecular Genetics, 2005, Vol. 14, No. 13 1763–1773doi:10.1093/hmg/ddi183Advance Access published on May 11, 2005

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by loss of function mutations in the ABCC6 gene (7–9).Furthermore, we found that a frequent (founder) mutation inABCC6, R1141X (10), may be associated with a strongincrease in the prevalence of premature coronary arterydisease (5).

The gene ABCC6 encodes a protein of 1503 amino acids,which contains three membrane-spanning domains and twoATP-binding cassettes (ABC). On the basis of amino acidalignments, ABCC6 has been assigned to the subfamily ofmultidrug resistance proteins (MRP) (11,12). Members ofthe MRP family are known to transport drugs, toxic substancesor lipids across the cell membrane, which may lead to clinicalmultidrug resistance of cancer cells. The physiological sub-strate of ABCC6 is not yet known. However, Ilias et al. (13)found that glutathione conjugates, including leukotrien-C4(LTC4) and N-ethylmaleimide S-glutathione (NEM-GS), areactively transported by human ABCC6. In three ABCC6mutant forms associated with PXE, loss of ATP-dependenttransport activity was observed (13).

Expression of ABCC6 is high in liver and kidney (8,14). Theprotein is located at the basolateral side of hepatocytes (15).This location suggests that ABCC6 exports substances out ofthe liver cell into the blood stream. Interestingly, ABCC6mRNA expression is low in tissues usually affected by thedisease: skin, eye and blood vessels (8). This finding has ledto the hypothesis that PXE is a systemic disease (16). On theother hand, the protein can be detected in cultured fibroblasts,which could support a local origin of PXE pathology (10,17).

Mutation analysis of ABCC6 (10,18) and the in vitro trans-port studies with PXE-associated ABCC6 mutants (13) suggestthat PXE is primarily caused by deficiency in transport activityof this ABC transporter. Yet, the exact role of ABCC6 in theaetiology of PXE is still unclear. To investigate the biologicalfunction of ABCC6, we constructed a genetically modifiedmouse in which Abcc6 was disrupted. Analysis of Abcc6deficient mice showed calcification of elastic fibres in

Bruch’s membrane in the eye and in blood vessel walls. Inaddition, alterations in plasma HDL cholesterol and plasmacreatinine, but not in mineral balance, were found. Conse-quently, the Abcc6 deficient mouse appears to be a suitableanimal model for further studies of PXE.

RESULTS

Generation of Abcc62/2 mice

The targeting strategy for generating mice lacking Abcc6 isshown in Figure 1A. We chose to delete the coding sequenceof the first nucleotide-binding fold (NBF1), because PXEcausing mutations occur in this region (18,19). In addition,in vitro studies showed that NBFs are essential for the functionof ABC proteins in general (12) and ABCC6 in particular(12,13). Finally, this strategy has been previously successfulin creating functional knock-out mice for other ABCCtransporters, such as MRP1 (20).

The largest part of Abcc6 NBF1, including the Walker Aand B and signature C motifs, is encoded in exons 16, 17and 18. In the targeting construct, the genomic fragment con-taining these exons was replaced by a selectable hygromycinresistance marker gene (Fig. 1). After electroporation of theconstruct into ES cells, hygromycin-resistant clones werescreened for homologous recombination. Out of 96 ES cellclones examined, only nine clones showed correct integrationof the construct at the targeted site as indicated by Southernanalysis using probes located external to the construct(Fig. 1) and by PCR using primers located in the undeletedpart of exon 18 and in the hygromycin resistance gene (datanot shown). ES cells of correctly targeted clones were injectedinto C57Bl/6 blastocysts, resulting in chimeric mice that trans-mitted the targeted allele, as detected with Southern blotanalysis and PCR. Heterozygous offspring were mated withC57Bl/6 mice, for backcrossing to C57Bl/6, and were

Figure 1. Targeted disruption of the NBF1 of mouse Abcc6. (A) Schematic representation of the wild-type mouse Abcc6 locus containing exons 14–22, thetargeting construct and the targeted locus. In the wild-type allele, restriction sites are indicated, which were used to make the targeting construct. The targetingconstruct contains an anti-sense hygro cassette replacing exons 16, 17 and most of 18. These exons encode the largest part of NBF1. In the targeted allele, thepositions of 50 and 30 probes for Southern analysis are indicated with bars. These are external to the construct and were used to verify correct genomic integrationof the construct. Restriction sites and expected fragments lengths in the targeted allele are shown. (B) Southern blot analysis identifying wild-type and targetedalleles in wild-type (þ/þ) and heterozygous (þ/2) ES cell clones.

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interbred to generate Abcc6þ/ þ , Abcc6þ/ 2 and Abcc62/2

mice. Both Abcc6þ/ 2 and Abcc62/2 mice were viable andfertile. Inheritance of the mutated allele showed no deviationfrom the expected Mendelian ratio.

Confirmation of disruption of Abcc6

We analyzed mRNA and protein expression of the targetedlocus. Abcc6 mRNA expression was first studied byRT–PCR on Abcc62/2 mouse liver cDNA with threedifferent primers sets with amplicons located up- and down-stream of the targeted area and within the targeted area.Using cDNA from Abcc62/2 mouse liver, no product wasamplified with the primer set located within the deleted area,but the amplicons up- and down-stream of the deletionwere present. This indicates that an aberrant Abcc6 transcriptis formed, lacking the targeted NBF1 region. Next, wesequenced a 2 kb RT–PCR product between exons 15 and19, spanning the targeted area. The latter showed that theopen reading frame, in fact any putative reading frame, wasinterrupted by several stop codons through introduction ofthe anti-sense hygromycin cassette in the transcript.

To study Abcc6 protein expression, we raised ratmonoclonal antibodies (Mabs) against a fusion proteincontaining amino acids 843–1000 of mouse Abcc6. TwoMabs (M6II-24 and M6II-68) were used in western blotswith protein fractions derived from liver and kidney ofwild-type and Abcc62/2 mice (Fig. 2). In the wild-typefractions, both Mabs stained a protein band at 165 kDa,which corresponds to the predicted size of full-length mouseAbcc6. Abcc6 protein was not detected in the protein fractionsof Abcc62/2 mice (Fig. 2).

Phenotype of Abcc62/2 mice

To find out whether Abcc6 deficient mice spontaneouslydevelop pathology, we kept Abcc62/2 and wild-type miceunder standard conditions in our stables and checked themregularly. The oldest Abcc62/2 mice analyzed were 22months old and no gross abnormalities or a clear differencein mortality with wild-type littermates was seen. Mice ofvarious ages were sacrificed for histological examination.

Abcc62/2 mice spontaneously developed calcification ofblood vessels. This phenomenon was most prominent in thecortex of the kidney (Fig. 3). Here, calcification of bloodvessels first appeared �6 months and progressed withage (Table 1). Both male and female Abcc62/2 mice wereaffected. In medium-sized arteries, mineral deposits werenoticed within the vessel wall (Figs 3 and 4A and E). Elasticastaining in adjacent sections revealed that calcium was deposi-ted along the elastic fibres in the vessel wall (Fig. 4B and D).Smaller blood vessels and capillaries were extensively calci-fied with deposits protruding into the lumen (Figs 3 and 4F).

In older Abcc62/2 mice, blood vessel calcification was alsofound in other organs. In 17–19-month-old Abcc62/2 mice,calcium deposits were seen in small blood vessels in manytissues examined, such as adipose tissue at various topo-graphic locations, skin, submandibular gland and the tongue.In addition, calcium deposition was observed in the elastic

lamellae of the aorta, in the elastic fibres of the vena cava(Fig. 5A and B) and in the internal elastic lamina of coronaryarteries. Examination of these tissues in age-matchedwild-type mice revealed no calcifications.

As skin and eye are usually the first organs to show clinicalsigns in patients, we examined these structures extensively inAbcc62/2 mice. The mice had no skin abnormalities that werevisible to the naked eye. Light microscopic analysis of the skinof the neck, arm pit, groin and back showed some sparse cal-cified blood vessels, but calcium deposition at dermal elasticfibres was not observed. Next, we employed electronmicroscopy on the skin of the groin of 18-month-old threeAbcc62/2 and three wild-type mice. No clear differenceswere seen in the dermal elastic fibres, although these fibresin Abcc62/2 mice sometimes had a mottled appearance(data not shown).

In the eyes of old Abcc62/2 mice, calcification of Bruch’smembrane was demonstrated with Alizarin red S staining(Fig. 5D). With electron microscopy, we observed accumu-lation of electron dense material in the lamina elastica ofBruch’s membrane of Abcc62/2 mice (Fig. 6). In addition,abnormal fibres were seen having an electron lucent core anda very electron dense cortex. These fibres often branched andran crisscross in the lamina elastica, the inner collagenouszone and, to lesser extent, in the outer collagenous zone ofBruch’s membrane. These abnormal structures were found inall three of the 18-month-old Abcc62/2 mice examined, butwere not observed in the age-matched wild-type mice (n ¼ 3).

Immunolocalization of Abcc6

Abcc6 protein expression and localization was studied usingthe anti-Abcc6 Mabs on frozen sections of several tissues ofwild-type and Abcc62/2 mice. In the liver of wild-typemice, clear Abcc6 staining was observed in the basolateralmembranes of the hepatocytes. In the kidney of wild-type

Figure 2. Western blot stained for Abcc6 using Mab M6II-68. Protein frac-tions were derived from liver and kidney of wild-type and Abcc62/ 2 mice.The predicted size of mouse Abcc6 is 165 kDa and a band corresponding tothis size is present in wild-type animals, but absent in Abcc62/ 2 animals.In addition, the antibody recognizes a band at 90 kDa in wild-type liver frac-tions, which is not present in Abcc62/ 2 mice. The nature of this protein bandis not yet known, but it may represent a proteolytic fragment or a splice variantof Abcc6.

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mice, the Mabs localized the protein to the basal membranesof the proximal tubules. Negative control stainings, usingeither an irrelevant, isotype matched antibody or frozentissue sections from Abcc62/2 mice, showed no staining(Fig. 7).

As PXE patients develop pathology in skin, eye and bloodvessels, we examined these structures for the presence ofAbcc6 protein in wild-type and Abcc62/2 mice. No immunor-eactivity for Abcc6 was seen in blood vessels of the kidney,which, as mentioned earlier, can develop pathology in theAbcc62/2 mouse. Eye and skin also did not show Abcc6 immu-noreactivity (Fig. 7). In addition, we examined tissues ofnewborn mice to see whether Abcc6 protein was developmen-tally expressed at these pathogenic sites: no Abcc6 proteinwas detected in skin, eye and renal blood vessels in 0- and3-day-old wild-type and Abcc62/2 mice (data not shown).

Analysis of blood plasma

In view of a putative systemic origin of PXE (16), we analyzedseveral potentially relevant constituents of blood plasma of11-month-old Abcc62/2 mice and healthy, age-matched

Figure 3. Kidney sections of Abcc62/ 2 (A) and wild-type (B) mice of 17 months. von Kossa staining shows extensive calcification (black staining) of bloodvessels in the cortex of Abcc62/2 kidney. Calcium deposits protrude into the lumen of small blood vessels. Larger arteries show calcification within the vesselwall. Bar, 100 mm.

Table 1. Number of calcified blood vessels per longitudinal kidney section

Age (months) Wild-type (n ) Abcc62/ 2 (n )

2.5 0 (5) 0 (4)6 0 (4) 0.9 + 0.5 (4)10 0 (3) 4.6 + 2.5 (3)17 0.1 + 0.2 (5) 25.2 + 18.0 (5)

n, number of mice. Per mouse, three von Kossa stained kidney sectionswere analysed and the counts were averaged. Mean + SD.




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control mice. Interestingly, no significant changes were foundin plasma mineral levels (Table 2). However, statistically sig-nificant differences were found in HDL-cholesterol levels,which were reduced in Abcc62/2 mice (P ¼ 0.01), and in crea-tinine levels, which were elevated in the knock out (P ¼ 0.03),when compared with wild-type mice. To confirm and extendthese findings, we analyzed plasma lipid spectrum and plasmalevels of creatinine and urea of 2.5- and 8-month-old mice,which were fasted 7–9 h prior to collection of blood(Table 3). In 8-month-old mice, the significant changes inHDL cholesterol and in creatinine levels were reproduced. Inyoung animals, however, no difference with wild-type micewas found. Plasma urea levels did not differ at these ages.

DISCUSSION

When ABCC6 was identified in the year 2000 as the genecausing PXE, this came as a surprise since the relationbetween this ABC transporter and elastic fibre calcificationwas not readily apparent (7–9). We now disrupted Abcc6in the mouse to further investigate this relationship. Themost important finding is that Abcc62/2 mice spontaneouslydeveloped calcification and elastic fibre abnormalities inBruch’s membrane and blood vessel walls. This phenotyperesembles the ophthalmic and cardiovascular pathologies ofPXE (3) and provides us with a suitable animal model forfurther investigation of the aetiology of PXE.

Figure 4. Kidney sections of 10-month-old (A) and 17-month-old (B–F) Abcc62/ 2 mice, stained with von Kossa (A and B), HE (C, E and F) and elastica,which stains elastic fibres black (D). Calcium deposits (arrows in E and F) are found in medium-sized arteries (A and E) and capillaries, close to the glomerulus(arrow in F). Calcium deposits are localized along the elastic fibres of the lamina elastica interna (B–D). Bars, 50 mm (A) and 20 mm (B–F).




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Phenotype of Abcc62/2 mice partly resembles PXEpathology

Progressive calcification and fragmentation of elastic fibres inskin, eye and blood vessels are characteristics for human PXE(2,3,21). The phenotype of the Abcc62/2 mouse partlyresembles the pathology of PXE patients: Abcc62/2 micespontaneously developed calcification and elastic fibre abnor-malities in blood vessels and Bruch’s membrane in the eye,whereas no clear changes were seen in the extracellularmatrix of the skin.

In the Abcc62/2 mouse, calcification apparently affectedprimarily small arteries, with small arteries in the cortex ofthe kidney being most vulnerable. Calcium deposits could bedetected at the elastic component in the vessel wall. In oldAbcc62/2 mice, large blood vessels were also affected, as

exemplified by calcification of the elastic lamellae of theaorta and elastic fibres of the vena cava.

These features correspond well to the pathology in PXEpatients. Intermittent claudication, which is due to mineraldeposits in arteries in the legs, is a common cardiovascularsymptom of PXE (2,3). In addition, calcification of arteriesin internal organs of PXE patients has been demonstrated byvarious techniques such as radiography and echography. Inview of the kidney pathology in the Abcc62/2 mouse, it isof interest that 25% of PXE patients develop renovascularhypertension and echographic opacities due to calcificationsof arteries in kidneys, spleen and pancreas (3,22–24).Finally, there are histopathologic data of blood vessel calci-fication in various organs of PXE patients. Recently, a largenumber of tissues from two PXE patients were examinedultrastructurally and alterations of the elastic component wereseen in almost all small- and medium-sized vessels in allorgans. In addition, fragmentation and mineralization of elasticfibres were observed in the aorta and vena cava (4).

In Bruch’s membrane of old Abcc62/2 mice, calcificationwas found by employing light microscopy and Alizarin redS staining. At the ultrastructural level, we observed electrondense material in the lamina elastica of Bruch’s membrane.In addition, abnormal fibres with an electron dense cortexwere present in Bruch’s membrane. Similar fibres have beendescribed as calcified elastic fibres in ageing human Bruch’smembrane (25). As we did not see these abnormalities inage-matched control mice, this may resemble the calcificationprocess of Bruch’s membrane in PXE patients (3,4),eventually leading to breaks in Bruch’s membrane.

The skin of the Abcc62/2 mouse appears to be lessinvolved in the disease than is generally the case in PXEpatients. PXE patients frequently have extensive minerali-zation of elastic fibres in the dermis, typically in the neckand flexural areas (2). In contrast, in the Abcc62/2 mouse,von Kossa staining did not reveal calcification of elasticfibres in these skin areas, whereas in the same tissue, calcifica-tion of blood vessels had already occurred. The reason for thisdifference between man and mice is not clear. However, inPXE patients, the relative involvement of eye, skin and cardio-vascular system varies considerably, even between familymembers having the same mutations (3,18). This suggeststhat environmental factors or other genetic factors may havean impact on the expression of the disease (26), which mayshift the balance towards a cardiovascular and eye phenotypein the mouse.

Localization of Abcc6

An important unresolved issue is whether PXE is a systemicdisease (16). The alternative is that it is caused by local dys-function of the protein within the connective tissue itself,e.g. in fibroblasts (4,17). When examining ABCC6 expressionin human tissues, only low levels of ABCC6 mRNA werefound in tissues frequently affected by PXE (8) and noABCC6 protein was detected by immunohistochemistry inskin and eye (15). In contrast, ABCC6 mRNA and proteinwere highly expressed in human liver, which is unaffectedby PXE (8,14). Beck et al. (27) examined mouse tissueswith polyclonal antibodies and found a more widespread

Figure 5. Calcification in 17–19-month-old Abcc62/ 2 mice, demonstratedby von Kossa staining (A–C) and Alizarin red S (D). (A) Calcium deposition(black staining) in lamina elastica interna of the aorta dorsalis. (B) Calcifica-tion (black staining) of elastic fibres in the wall of the vena cava, shown inmore detail in (C). (D) Alizarin red S staining shows calcification (redstaining) in Bruch’s membrane (between arrowheads). Bars, 50 mm (A andB) and 20 mm (D).




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Abcc6 protein expression in multiple tissues including liver,kidney, eye, skin and blood vessels. In the present study,we used polyclonal and monoclonal antibodies to localizeAbcc6, with Abcc62/2 mouse tissues as negative control. Inthe mouse, we found essentially the same localization as pre-viously reported for the human tissues (15): Abcc6 was loca-lized to the basolateral membranes of hepatocytes and basalmembranes of the proximal tubules of the kidney. Further-more, examining tissue of adult as well as newborn mice,we could not detect Abcc6 protein at pathogenic sites suchas renal blood vessels, the skin or Bruch’s membrane in theeye. Therefore, our data do not support a local but a systemicaetiology of PXE in the mouse.

Analysis of blood plasma

The high expression of Abcc6 in liver and kidney (8,14,28), itsputative function as transporter and its localization to baso-lateral membranes of hepatocytes (15) are ideal for a potentialsystemic origin of PXE by changes in blood composition.Analysis of blood plasma resulted in three important findings.

First, the plasma concentration of minerals, such as calcium,was not changed in the Abcc62/2 mouse. Therefore, nosupport was obtained for a systemic disturbance of the electro-lyte balance as systemic cause of PXE. This is particularlyrelevant as several studies suggested that mineral intake andbalance may influence the PXE disease state (24,29,30).

Secondly, creatinine levels were significantly increased inolder Abcc62/2 mice (�8 months), which points towardimpaired kidney function (31). This is an interesting finding,since, in patients with chronic kidney disease and renalfailure, increased calcification as well as thickening andelastic changes of the blood vessel wall have been reported(32). These changes may be similar to those found in thecarotid artery of PXE patients (33). In this way, kidneydysfunction could not only be a consequence of generalizedvascular disease in PXE but also a contributor.

Thirdly, plasma HDL cholesterol was reduced by 25% inAbcc62/2 mice of �8 months. This is remarkable becauselow HDL-cholesterol levels are associated with vascularpathology. In the literature, there is also support for theinvolvement of abnormal serum lipid levels in PXE. In afamily with a proven apoa1/CIII deficiency of the Detroittype, some striking clinical similarities with PXE have beenreported (34). Cross-sectional studies of PXE patients and con-trols suggested depressed plasma HDL cholesterol (2) andhypertriglyceridemia in PXE (2,29). Recently, a DNA sequencepolymorphism in ABCC6 was found associated with alteredplasma HDL cholesterol and triglyceride levels (35).

The observed HDL reduction in Abcc62/2 mice of�8 months may be the result of the impairment of kidneyfunction. In patients with chronic renal failure, hepatic ApoA-I synthesis decreases and HDL levels fall, whereas plasmalevels of triglycerides rise (36,37). Alternatively, it is temptingto speculate that Abcc6, like many other ABC transporters(38), can be directly involved in lipid transport or metabolism.Although it is well known in humans that lipid distribution andcomposition changes considerably during development andageing (39), very little is known about an age-dependent regu-lation of Abcc6 expression (40), which could be a cause ofreduction of HDL in older mice.

In summary, we observed three, probably related, patho-logical events in the ageing Abcc62/2 mouse. At young age(2.5 months), calcification of renal vessel walls (and othertissues) was absent and renal function was normal asevidenced by normal creatinine, urea and cholesterol plasmalevels. In 8–19-month-old mice, progressive calcification ofblood vessels and other tissues occurred, serum creatininelevels rose, suggesting renal failure, and HDL-cholesterollevels fell. It is currently not clear what exact mechanism(s)underlie this potential chain of events.

Combining our data on Abcc6 protein localization andblood plasma values with those of the literature, we suggestthat alterations in plasma (lipid) composition may be involved

Figure 6. Electron micrographs of Bruch’s membrane (between arrow heads) of Abcc62/ 2 (A) and wild-type (B) mice, aged 18 months. As distinct from thewild-type mouse, the Abcc62/ 2 mouse shows electron dense material in the lamina elastica and a meshwork of fibres with an electron dense cortex and electronlucent core. Bar, 500 nm.




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in the development of clinical features of PXE. In this respect,it is important to test lipid substrates in in vitro Abcc6 trans-port studies. In addition, further studies of plasma (lipid) com-position in PXE patients with defined molecular lesions inABCC6 are warranted, whereas the Abcc62/2 mouse seemsto be a good model for dietary studies.

MATERIALS AND METHODS

Generation of Abcc6 2/2 mice

Isogenic mouse Abcc6 genomic DNA was obtained byscreening a 129/Ola mouse bacteriophage artificial chromo-some (BAC) library (Incite) with mouse Abcc6 cDNAprobes. A BAC containing the Abcc6 gene was isolated(BAC no. A-962B4) and a 14.5 kb HindIII restriction fragmentwas subcloned into pGEM7 (Promega, Madison, WI, USA).This fragment spans the genomic region of exons 15–22and encompasses the coding sequence of the NBF1 (roughlyexons 16–18).

A gene-targeting construct was designed in which most ofthe NBF1 coding sequence was replaced by a marker geneto select for hygromycin resistance (Fig. 1). The targetingvector was constructed on the basis of pBluescript II KS(þ/2) phagemid with a hygromycin resistance gene drivenby the mouse phosphoglycerate kinase promoter (PGK-hygrovector, kindly provided by Dr J. Wijnholds). A fragment of4.1 kb surrounding exon 19 and containing part of exon 18was cut out of genomic Abcc6 with Eco47III and BssHII

and was inserted into the PGK-hygro vector, to serve as the30 homologous arm of the targeting construct. Next, a 2.5 kbfragment surrounding exon 15 was isolated after digestionwith Pst I and Xba I and ligated to the PGK-hygro cassetteas the 50 arm. Sequencing of the final targeting constructshowed that exons 16, 17 and part of 18 were replaced bythe PGK–hygromycin sequence, which was oriented in anti-sense direction. Correct targeting would delete the codingsequence for amino acids 647–782 of Abcc6 protein. Thiseliminates the largest part of NBF1 including the Walker Aand B and the signature C motifs.

The 12.5 kb targeting fragment was linearized by BssHIIand transfected into 129/OLA-derived E14 ES cells byelectroporation. Hygromycin resistant clones were pickedand expanded. After genotyping, the karyotype of ES cellsof correctly targeted clones was checked. ES cells wereinjected into C57Bl/6 blastocysts to generate chimeric malemice, which were mated with C57Bl/6 females. Offspringheterozygous for the disrupted allele (Abcc6þ/2) were matedwith C57Bl/6 mice, for backcrossing to C57Bl/6, and wereinterbred to generate Abcc6þ/þ, Abcc6þ/ 2 and Abcc62/2

mice. Most data of the present study were obtained on miceof backcross number 2 to C57Bl/6. In addition, Abcc62/2

and wild-type mice of backcross number 5 to C57Bl/6 wereused in experiments to confirm renal blood vessel calcificationin Abcc62/2 mice and in experiments analyzing blood compo-sition of 2.5- and 8-month-old mice. Mice were kept in a 12 hlight (,100 lux)/12 h dark cycle and had free access to waterand food.

Genotyping

Genotyping was done with Southern blot and multiplex PCR.ES cells and the first generation of mice were genotyped byboth Southern blot and PCR. After confirmation of the geno-type and correct chromosomal integration, next generationsof mice were genotyped only by PCR.

DNA was isolated from ES cells and snippets of the ears,according to standard protocols (41). Multiplex PCRemployed the following primers: 18F (50-TGA-ATC-TTT-CTG-GGG-GCC-AG-30), 18R (50-GTA-CCC-TGG-AGC-AAT-CCA-CT-30) and T2 (50-ATG-TGG-AAT-GTG-TGC-GAG-GCC-30). Primers 18F and 18R amplify a 163 bp fragmentof exon 18 on the wild-type allele, whereas primers 18R andT2 yield a 246 bp fragment spanning the boundary ofPGK-hygro cassette on the targeted allele. For Southernanalysis, genomic DNA was digested by EcoRI and Kpn I or

Figure 7. Immunohistochemical staining for Abcc6 (brown colour) using MabM6II-68 (C and D) and M6II-24 (A, B, E and F). In wild-type mice, Abcc6 isexpressed in the basolateral membranes of the hepatocytes of the liver (B) andin the basal membranes of the proximal tubules of the kidney (D). We detectedno Abcc6 immunoreactivity in skin (E) and eye (F), as illustrated here withsections of the skin of the back (E) and of the posterior segment of the eye(F) of albino wild-type mice. Arrow heads in (F) point at Bruch’s membrane.Tissue from Abcc62/ 2 mice was used as negative control: no immunoreactiv-ity was detected in liver and kidney of Abcc62/ 2 mice (A and C).Counterstaining was done with haematoxylin.

Table 2. Plasma mineral levels in 11-month-old Abcc62/ 2 and wild-type mice

Wild-type (n ¼ 11) Abcc62/ 2 (n ¼ 14) t-test (P-value)

Sodium 151 + 2 151 + 3 0.72Calcium 2.66 + 0.07 2.66 + 0.11 0.94Phosphate 3.03 + 0.35 3.01 + 0.37 0.87Chloride 116 + 2 115 + 2 0.21Magnesium 1.13 + 0.10 1.09 + 0.08 0.38

Mean + SD in mM/l.




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by EcoRI and Bcl I (Biolabs), size fractionated on 0.8% agarosegels and transferred to GeneScreen Plus nylon membranes(NEN). Membranes were probed with radiolabelledPCR-amplified fragments of genomic Abcc6. The probes werelocated external to the construct (Fig. 1).

Generation of antibodies to mouse Abcc6

Antibodies against mouse Abcc6 were generated using theglutathione S-transferase (GST) gene fusion system (Pharma-cia). The third transmembrane domain of Abcc6 was amplifiedby PCR on mouse liver cDNA. This part of the protein islocated downstream of the targeted region. Primers (forward:50-GAGACCATGGGSGCCCTGGTGGGTCTT-30; reverse:50-GAGAATTCTTGGGGATGCGAGCGTAG-30) yielded an1190 bp amplicon, which was cloned into the pGEMTeasyvector (Promega). Digestion with Nco I resulted in a 471 bpfragment corresponding to amino acids 843–1000 of mouseAbcc6. This fragment was cloned into a derivative ofpGEX-3X plasmid (Pharmacia). After transformation ofEscherichia coli DH5a cells, the 44 kDa Abcc6–GST fusionprotein was produced, isolated and purified using glutathionebeads. Abcc6–GST fusion protein was injected in rabbitsand rats for production of polyclonal and monoclonal anti-bodies, respectively. Immunization and fusion protocols formonoclonal production were as described earlier (42,43). Inbrief, a 12-week-old female Wistar rat received �200 mgfusion protein per injection. Four booster injections weregiven. Draining lymph nodes and the spleen of the rat werefused with Sp20 mouse myeloma cells. Supernatants ofobtained hybridoma cells containing Mabs were screened onELISA plates coated with specific fusion protein and, as acontrol, on plates coated with irrelevant fusion protein.

Antibody binding was detected using HRP-labelled rabbit-anti-rat serum (1:500, Dako, Copenhagen, Denmark)and 5-amino-2-hydroxybenzoic acid (Merck, Darmstadt,Germany) and 0.02% H2O2 as a chromogen. Screening of hybri-doma supernatants yielded several positive hybridomas. TwoMabs, M6II-24 and M6II-68, were selected for further analysis.Isostrips (Serotec, Oxford, UK) showed that M6II-24 is ofIgG2a subclass and M6II-68 is of IgG2b subclass.

Immunodetection of Abcc6

For Western blot analysis, liver and kidney samples werethawed in lysis buffer, consisting of 50 mM Tris–HCl, pH7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 mM

PMSF, 1 mM Na3VO4 and protease inhibitor cocktail(Roche). Next, the tissue was pottered, kept on ice for30 min and cleared by centrifugation at 10 000g for 15 min.Protein concentration was determined by micro-BCA proteinassay reagent kit (Pierce). Protein samples (30 mg/lane) weresize fractionated by SDS–polyacrylamide (7.5%) gel electro-phoresis and transferred onto nitrocellulose membranes byelectroblotting. Membranes were probed with anti-Abcc6Mabs M6II-24 and M6II-68, described earlier, and stainedusing HRP-labelled donkey-anti-rat secondary antibodies(Jackson Immunoresearch) and a chemiluminescent detectionsystem (Pierce).

Immunolocalization of Abcc6 was done on cryosections(4–6 mm). These were air dried overnight and fixed for7 min in acetone at room temperature. Slides were incubatedwith primary antibody for 1 h at room temperature.HRP-labelled goat-anti-rat serum (1:100, Santa Cruz, CA,USA) was used as secondary reagent. Colour developmentwas with 0.4 mg/ml amino-ethyl-carbazole (AEC) and0.02% H2O2 as a chromogen.

Tissue collection and morphological analysis

Mice were sacrificed by CO2/O2 and cervical dislocation. Forimmunohistochemistry and biochemical analysis, tissues wereexcised, frozen in liquid N2 and stored at 2808C until use.

For routine histological analysis, organs were excised, fixedin 4% paraformaldehyde in 0.1 M phosphate buffer for at least15 h, dehydrated, embedded in paraffin and sectioned in4–7 mm thick slices. We used standard staining proceduresof haematoxylin–eosin (HE), von Kossa, Alizarin red S,Elastica von Gieson (EVG) and Elastica staining. Organsexamined included liver, kidney, skin (of groin, neck andback), eye, heart, descending aorta, lung, stomach, tongue,lower lip and ear. For orientation, eyes were marked nasallywith Alcian blue (5 in 96% ethanol). Ages of the mice were6, 10, 11, 14, 17 and 19 months. Per genotype (Abcc62/2

and wild-type) and per age group, 3–5 mice were examined.In addition, 18-month-old mice were fixed and prepared for

electron microscopy. They were anesthetized by intraperito-neal injection with Nembutal and perfused through the heartwith 1% glutaraldehyde and 1.25% paraformaldehyde in0.1 M cacodylate buffer, pH 7.4. Eye, skin, aorta and liverwere post-fixed in 1% OsO4 in 0.1 M cacodylate buffer, pH7.4, and embedded in epon for electron microscopic analysis.Ultra-thin sections were stained with lead citrate and uranyl

Table 3. Plasma lipid, creatinine and urea levels in 2.5- and 8-month-old Abcc62/ 2 and wild-type mice

2.5-month-old mice 8-month-old mice

Wild type (n ¼ 8) Abcc62/ 2 (n ¼ 6) t-test (P-value) Wild-type (n ¼ 8) Abcc62/ 2 (n ¼ 8) t-test (P-value)

Total cholesterol (mM/l) 2.2 + 0.6 2.4 + 0.9 0.61 3.2 + 0.7 2.3 + 0.7 0.01HDL-cholesterol (mM/l) 2.3 + 0.5 2.4 + 0.7 0.86 3.0 + 0.2 2.2 + 0.7 0.01Triglycerides (mM/l) 0.46 + 0.18 0.42 + 0.18 0.68 0.62 + 0.17 0.49 + 0.145 0.13Creatinine (mM/l) 8.75 + 1.3 8.67 + 0.5 0.87 7.8 + 1.3 9.3 + 0.7 0.01Urea (mM/l) 7.2 + 1.0 6.9 + 1.3 0.64 8.1 + 1.8a 8.4 + 0.9a 0.71a

aThese urea values were determined in five wild-type and six Abcc62/ 2 mice.




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acetate. Other tissues of these mice were embedded in paraffinand examined at the light microscopical level.

For blood analysis, Abcc62/2 and wild-type mice weresacrificed by CO2/O2 and blood was collected in tubes con-taining lithium heparin. For measurement of the lipid spec-trum, mice were fasted for 7–9 h prior to collection of blood.

Statistical analysis

Statistical analysis was carried out using Student’s t-test,two-sided.

ACKNOWLEDGEMENTS

The authors wish to thank Dr J. Wijnholds for excellent assis-tance in the course of creating the knockout mouse, ProfessorDr R.S. Reneman, Dr M. Tripp and Dr A. Plomp for helpfuldiscussions, Ing. I. Versteeg and Ing. A.J.N. Schoonderwoerdfor technical assistance. Financial support of the StichtingBlindenpenning and the Algemene Nederlandse Verenigingter Voorkoming van Blindheid is gratefully acknowledged.

Conflict of Interest statement. None declared.

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Disruption of Abcc6 in the mouse: novel insight in the pathogenesis of pseudoxanthoma elasticum

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